Characterization and expression analysis of the groESL operon of Bartonella bacilliformis

The groESL operon of Bartonella bacilliformis, a facultative intracellular, Gram-negative bacterium and etiologic agent of Oroya Fever, was characterized. Sequence analysis revealed an operon containing two genes of 294 (groES) and 1632 nucleotides (groEL) separated by a 55-nt intergenic spacer. The operon is preceded by a 72-nt ORF (ORF1) that encodes a hypothetical protein with homology to a portion of the HrcA repressor for groESL. A divergent fumarate hydratase C (fumC) gene lies further upstream. Deduced amino acid sequences for B. bacilliformis GroEL and GroES revealed a high degree of identity with homologues from other Bartonella and alpha-Protebacteria. A single transcriptional start site (TSS) was mapped 79 nucleotides upstream of the groES start codon, regardless of incubation temperature. The TSS was located immediately 5\u27 to a potential controlling inverted repeat of chaperonin expression (CIRCE) element and is preceded by a sigma70-like promoter. The operon is followed by a predicted rho-independent transcriptional terminator. Northern blot analysis indicated that groES and groEL are co-transcribed as a single mRNA of approximately 2.4 kb. A 6-h time course analysis by qRT-PCR showed that groEL expression increases 1.3-fold within 30 min of a temperature upshift from 30 to 37 degrees C, with maximum transcription reached after 60 min (approximately 4.3-fold), followed by a steady decrease to background (30 degrees C) transcription levels by 6 h. Western blot analysis revealed a 1.4- and 1.5-fold increase in GroEL synthesis following a temperature upshift or by inhibiting DNA supercoiling with coumermycin A1, respectively. Functional expression and complementation of temperature-sensitive Escherichia coli groES or groEL mutants with the cloned operon allowed them to grow at otherwise restrictive temperatures

Protection Against Marburg Virus Using a Recombinant VSV-Vaccine Depends on T and B Cell Activation

Marburg virus (MARV) is the causative agent of hemorrhagic fever outbreaks with high case fatality rates. Closely related to Ebola virus, MARV is a filamentous virus with a negative-sense, single-stranded RNA genome. Although extensive studies on filovirus countermeasures have been conducted, there are no licensed treatments against MARV infections. An experimental vaccine based on the recombinant vesicular stomatitis virus (VSV) expressing the MARV-Musoke glycoprotein demonstrated complete protection when a single dose was administered 28 days and up to 14 months prior to MARV challenge. Here, we analyzed the protective efficacy of an updated vaccine expressing the MARV-Angola glycoprotein (VSV-MARV). A single dose of VSV-MARV given 5 weeks before challenge provided uniform protection with no detectable viremia. The vaccine induced B and T cell proliferation and, importantly, antigen-specific IgG production. Transcriptomic signatures confirm these findings and suggest innate immunity engendered by VSV-MARV may direct the development of protective humoral immunity

UK B.1.1.7 variant exhibits increased respiratory replication and shedding in nonhuman primates.

The continuing emergence of SARS-CoV-2 variants calls for regular assessment to identify differences in viral replication, shedding and associated disease. In this study, African green monkeys were infected intranasally with either a contemporary D614G or the UK B.1.1.7 variant. Both variants caused mild respiratory disease with no significant differences in clinical presentation. Significantly higher levels of viral RNA and infectious virus were found in upper and lower respiratory tract samples and tissues from B.1.1.7 infected animals. Interestingly, D614G infected animals showed significantly higher levels of viral RNA and infectious virus in rectal swabs and gastrointestinal tract tissues. Our results indicate that B.1.1.7 infection in African green monkeys is associated with increased respiratory replication and shedding but no disease enhancement similar to human B.1.1.7 cases. ONE-SENTENCE SUMMARY: UK B.1.1.7 infection of African green monkeys exhibits increased respiratory replication and shedding but no disease enhancement

A single intranasal dose of chimpanzee adenovirus-vectored vaccine protects against SARS-CoV-2 infection in rhesus macaques

The deployment of a vaccine that limits transmission and disease likely will be required to end the coronavirus disease 2019 (COVID-19) pandemic. We recently described the protective activity of an intranasally administered chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike (S) protein (ChAd-SARS-CoV-2-S [chimpanzee adenovirus-severe acute respiratory syndrome-coronavirus-2-S]) in the upper and lower respiratory tracts of mice expressing the human angiotensin-converting enzyme 2 (ACE2) receptor. Here, we show the immunogenicity and protective efficacy of this vaccine in non-human primates. Rhesus macaques were immunized with ChAd-Control or ChAd-SARS-CoV-2-S and challenged 1 month later by combined intranasal and intrabronchial routes with SARS-CoV-2. A single intranasal dose of ChAd-SARS-CoV-2-S induces neutralizing antibodies and T cell responses and limits or prevents infection in the upper and lower respiratory tracts after SARS-CoV-2 challenge. As ChAd-SARS-CoV-2-S confers protection in non-human primates, it is a promising candidate for limiting SARS-CoV-2 infection and transmission in humans

Novel neutralizing monoclonal antibodies protect rodents against lethal filovirus challenges

Filoviruses are the causative agents of lethal hemorrhagic fever in human and non-human primates (NHP). The family of Filoviridae is composed of three genera, Ebolavirus, Marburgvirus and Cuevavirus. There are currently no approved vaccines or antiviral therapeutics for the treatment of filovirus infections in humans. Passive transfer of neutralizing antibodies targeting the Ebola virus (EBOV) glycoprotein (GP) has proven effective in protecting mice, guinea pigs and NHP from lethal challenges with EBOV. In this study, we generated two neutralizing monoclonal antibodies (MAbs), termed S9 and M4 that recognize the GP of EBOV or multiple strains of Marburg virus (MARV), respectively. We characterized the putative binding site of S9 as a linear epitope on the glycan cap of the GP1 subunit of the EBOV-GP. The M4 antibody recognizes an unknown conformational epitope on MARV-GP. Additionally, we demonstrated the post-exposure protection potential of these antibodies in both the mouse and guinea pig models of filovirus infection. These data indicate that MAbs S9 and M4 would be good candidates for inclusion in an antibody cocktail for the treatment of filovirus infections

Immunological correlates of protection afforded by PHV02 live, attenuated recombinant vesicular stomatitis virus vector vaccine against Nipah virus disease

IntroductionImmune correlates of protection afforded by PHV02, a recombinant vesicular stomatitis (rVSV) vector vaccine against Nipah virus (NiV) disease, were investigated in the African green monkey (AGM) model. Neutralizing antibody to NiV has been proposed as the principal mediator of protection against future NiV infection.MethodsTwo approaches were used to determine the correlation between neutralizing antibody levels and outcomes following a severe (1,000 median lethal doses) intranasal/intratracheal (IN/IT) challenge with NiV (Bangladesh): (1) reduction in vaccine dose given 28 days before challenge and (2) challenge during the early phase of the antibody response to the vaccine.ResultsReduction in vaccine dose to very low levels led to primary vaccine failure rather than a sub-protective level of antibody. All AGMs vaccinated with the nominal clinical dose (2 × 107 pfu) at 21, 14, or 7 days before challenge survived. AGMs vaccinated at 21 days before challenge had neutralizing antibodies (geometric mean titer, 71.3). AGMs vaccinated at 7 or 14 days before challenge had either undetectable or low neutralizing antibody titers pre-challenge but had a rapid rise in titers after challenge that abrogated the NiV infection. A simple logistic regression model of the combined studies was used, in which the sole explanatory variable was pre-challenge neutralizing antibody titers. For a pre-challenge titer of 1:5, the predicted survival probability is 100%. The majority of animals with pre-challenge neutralizing titer of ≥1:20 were protected against pulmonary infiltrates on thoracic radiograms, and a majority of those with titers ≥1:40 were protected against clinical signs of illness and against a ≥fourfold antibody increase following challenge (indicating sterile immunity). Controls receiving rVSV-Ebola vaccine rapidly succumbed to NiV challenge, eliminating the innate immunity stimulated by the rVSV vector as a contributor to survival in monkeys challenged as early as 7 days after vaccination.Discussion and conclusionIt was concluded that PHV02 vaccine elicited a rapid onset of protection and that any detectable level of neutralizing antibody was a functional immune correlate of survival

A novel model of lethal Hendra virus infection in African green monkeys and the effectiveness of ribavirin treatment

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are emerging zoonotic paramyxoviruses that can cause severe and often lethal neurologic and/or respiratory disease in a wide variety of mammalian hosts, including humans. There are presently no licensed vaccines or treatment options approved for human or veterinarian use. Guinea pigs, hamsters, cats, and ferrets, have been evaluated as animal models of human HeV infection, but studies in nonhuman primates (NHP) have not been reported, and the development and approval of any vaccine or antiviral for human use will likely require efficacy studies in an NHP model. Here, we examined the pathogenesis of HeV in the African green monkey (AGM) following intratracheal inoculation. Exposure of AGMs to HeV produced a uniformly lethal infection, and the observed clinical signs and pathology were highly consistent with HeV-mediated disease seen in humans. Ribavirin has been used to treat patients infected with either HeV or NiV; however, its utility in improving outcome remains, at best, uncertain. We examined the antiviral effect of ribavirin in a cohort of nine AGMs before or after exposure to HeV. Ribavirin treatment delayed disease onset by 1 to 2 days, with no significant benefit for disease progression and outcome. Together our findings introduce a new disease model of acute HeV infection suitable for testing antiviral strategies and also demonstrate that, while ribavirin may have some antiviral activity against the henipaviruses, its use as an effective standalone therapy for HeV infection is questionable. Copyrigh

Hydroxychloroquine prophylaxis and treatment is ineffective in macaque and hamster SARS-CoV-2 disease models

open access articleWe remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human COVID-19 clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies to help guide decisions. Here we assessed the prophylactic/ therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in 2 animal disease models. The standard human malaria HCQ prophylaxis (6.5 mg/kg given weekly) and treatment (6.5 mg/kg given daily) did not significantly benefit clinical outcome, nor did it reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. Similarly, when used for prophylaxis or treatment, neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Results from these 2 preclinical animal models may prove helpful in guiding clinical use of HCQ for prophylaxis/treatment of COVID-19

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo